Ideally one would be able to identify, and localize, biomolecules such as DNA and RNA throughout all the cells throughout a tissue, with nanoscale precision. Such mechanistic maps would reveal how epigenomic configurations and transcriptomic programs are configured to mediate cellular as well as organ-scale emergent functions, and pathologies. They would also provide systematic datasets that could enable generation of unbiased hypotheses that could be tested via causal perturbation, for a wide variety of basic and applied biological questions.
Current tools do not permit this. Optical methods maintain the spatial location of molecules, but the number of biomolecules that can be studied simultaneously is limited. On the other hand, transcriptomic approaches allow the multiplexed measurement of potentially all the RNA and DNA molecules, but spatial information is lost in the process. For example, in brain tissues all the current RNA sequencing methods involve grinding up or dissociating the neurons before sequencing, thereby destroying all spatial information about the cells in relation to the tissue. Moreover, the subcellular location of the sequences inside the individual cells is also lost, including all the information about the RNA contents of the axons, dendrites, and synapses, which is crucial for the understanding of neuronal communication.
International patent application serial number PCT/US15/16788, which is incorporated herein by reference and Chen et al., Science, 347, 543 (2015), teach that the resolution of conventional microscopy can be increased by physically expanding specimens, a process termed ‘expansion microscopy’ also referred to herein as “ExM”. The advantages to ExM include tissue clearing, resolution improvement, and higher tolerance to sectioning error due to the specimen expansion in the z-axis. In the ExM method, cultured cells, fixed tissue, or in principle other types of samples of interest, including biological materials, are infused with a composition, or chemical cocktail, that results in it becoming embedded in the sample material, and then the composition can be expanded isotropically, preferably with nanoscale precision, in three dimensions.
ExM physically magnifies tissues while preserving nanoscale isotropy. It would be desirable to leverage ExM to devise new methods for in situ sequencing of nucleic acids throughout all the cells in a tissue.